1. Field of the Invention
The instrument relates to conducting assays by kinetically measuring changes in light transmission through a plurality of samples contained in test tubes and, more particularly, the invention provides an inexpensive desktop instrument that may advantageously be used with a standard personal computer for conducting such assays.
2. Discussion of the Background
FIG. 1 is a perspective view of a prior art system including a personal computer 1 (IBM PC or PC-compatible), a printer 2 connected to the personal computer, a CRT monitor 3, and an instrument 4 known commercially as the LAL-4000. The LAL-4000 is specially adapted for performing an assay generally known as the LAL assay, which is a quantitative assay for bacterial endotoxins. Sample and a reagent are mixed in a disposable test tube 10 and incubated at 37.degree. C. No additional reagents, dyes or acids are required.
Prior to the development of the LAL-4000, the LAL assay was traditionally performed as a gel-clot test. In contrast, the assay as conducted using the LAL-4000 is a kinetic assay. The main distinction between the two methods is the manner in which the test is read. The gel-clot method is dependent upon the formation of a firm gel produced by the LAL components in response to the presence of endotoxins, while the kinetic turbidimetric method uses the increase in turbidity which preceeds gel formation as the basis for quantitation. Conducting the traditional gel-clot method depended upon reaching an end point of gel formation and required a substantial period of time to conduct, including a one hour incubation period. Interpreting the results involved a subjective judgment by the tester as to whether or not a gel had formed. In contrast, the kinetic assay provides a continuous measurement according to which the LAL-4000 measures the time required for samples to reach a specific level of turbidity that is achieved prior to the end point of gel formation. This time is referred to as the "onset time". The assay is essentially complete once the sample under test has attained an onset time or has incubated for a predetermined time period corresponding to a specific level of endotoxin.
A kinetic assay has several advantages over the end point gel-clot assay employing a fixed incubation period. These advantages include increased sensitivity, greater resolution, greater precision, the elimination of subjectivity in interpreting the results, and increased assay speed.
The LAL-4000 unit includes a fixed incubator block 6 in the form of a rectangular solid having a plurality of test tube wells 8 disposed in a rectangular array. The instrument was designed to use disposable test tubes of non-optical quality. A separate pulsed LED light source 12 and photodetector 14 (FIG. 2) are provided for each test tube, so that light from one of the LED's 12 shines through a sample in its associated test tube and impinges on the associated photodetector 14. Because a separate light source is used for each test tube (and therefore for each data channel), variability from one light source to the next presents problems in interpreting the collected data to obtain quantified results. The size and weight of the instrument 4 are increased because of the need to provide electric power to each of a plurality of light sources. Furthermore, because of the rectangular array in which the test tubes, light sources and photodetectors are disposed, it is difficult to service the photodetectors and light sources.
In the LAL-4000, each photodetector 14 corresponds to a data channel. The instrument handles the data channels independently, so that the measuring for each data channel may begin at a separate time. Each test begins when a test tube 10 is inserted into a well 8. Each detector 14 is placed lower than its corresponding light source, so that it does not receive light from the associated light source 12 unless a test tube is present, and the instrument relies on detecting light refraction through the lower portion of the test tube in order to determine that a test tube has been inserted. Because of the low quality and internal variability of the non-optical quality glass used in the disposable test tubes, the distorted light path through the test sample is sensitive to jarring. This problem is further compounded where, as in the LAL-4000, the optical path passes through the curved lower portion of the test tube, where the refracted light path is especially poor. At times, a significant period of time has elapsed before it has been discovered that the instrument had failed to detect the presence of a test tube and had not begun collecting data at the appropriate time.
Because the LAL-4000 is designed with the light path passing through the bottom of non-optical quality test tubes, the sensitivity of the instrument to vibration and jarring is increased. In addition, inherent variability in the spectral outputs of the separate light sources forces one to conduct a substantial amount of pre-screening of the components to be used. This added complexity detracts from the design of the LAL-4000.
The LAL-4000 provides twenty test tube wells 8, this number being expandable to forty by re-working the chassis at substantial cost.
Because of the physical configuration of the incubator block 6, the range of assays that may be conducted is limited to assays capable of using light at the wavelengths provided by the individual LED light sources. This is because of the impracticality of changing each light source and because of the inability to insert a light filter between each light source and each sample.
The LAL-4000 uses a commercially available power supply which provides +/-15VDC, +12VDC, and +5VDC regulated voltages. It further uses a commercial computer board set (four boards minimum: CPU, an analog to digital converter, an input/output board, and an analog interface). Additional circuit boards are required to sense and measure the changes in light transmission within the incubator. These separate boards are electronically connected with extensive wiring harnesses which are expensive to make and install and are prone to failure. The LAL-4000 is approximately twenty inches wide, eighteen inches deep and eight inches high. It has a volume of 3000 cubic inches and weighs 42 pounds.
The LAL-4000 communicates with its host computer through a standard RS-232 interface. It sends data to the host computer only when a predetermined change in light transmission is measured in a given well. This forces the data string to include the well identity and the time of the measured change, in addition to the actual magnitude of the change itself. Since the logic circuitry of the LAL-4000 determines what data is transmitted and when, the data is not in a predetermined, regular format, and its utility for a variety of purposes is diminished accordingly.
Shown in FIG. 2 is a diagrammatic top view of the incubator block 6 of the LAL-4000, showing the general locations of the test tube wells 8, the individual light sources 12 and the photodetectors 14. Also shown, in dashed lines, are isotherms 16. As noted above, a heater is provided to maintain the incubator block at substantially 37.degree. C. Accordingly, the center of the block is at a somewhat higher temperature than the edge portions of the block. Because of the rectangular array in which the test tubes 10 are disposed, there necessarily results a variation in temperature among the various samples under test. Such thermal gradients within the incubator have a detrimental effect on the assay. The temperature in a given well affects the speed at which the reaction takes place. Any such variablility affects the intercomparison of results. Accordingly, this variation further detracts from the reproducibility of the results.
The temperature variations within the incubator block also are experienced by the photodetectors 14. All photovoltaic devices have substantial temperature coefficients. For a given incident light, the current generated by the sensor 14 is a strong function of its ambient temperature. Accordingly, these temperature variations among the photodetectors even further adversely affect the reproducibility of the results.
Other instruments for making turbidimetric mesurements are known, including the Wako Toxinometer, Abbott Laboratories MS-2.RTM., General Diagnostics Coagamate, and a microplate reader, also called a micro-titer plate reader. The Toxinometer, the MS-2 and the microplate reader employ a rectangular grid of test tube wells or sample-containing cuvettes. Like the LAL-4000, the Toxinometer has relatively inaccessible electronic parts within the heater block and is a simple single-wavelength optical reader. Like the LAL-4000, the Toxinometer has wells arranged in a grid. Consequently, attempts to control the incubator temperature result in strong temperature gradients.
Like the LAL-4000, the Toxinometer and the MS-2 each have a separate light source for each sample well. Other instruments, including the Coagamate, employ a circular geometry, a single light source, and light detecting means which are not dedicated to a particular sample.
The Coagamate uses a carousel. This requires the use of optical grade cuvettes, because the optical path through a disposable tube on a moving carousel cannot be consistently established on a repeatable basis. Furthermore, a moving carousel is difficult to incubate because of poor heat transfer to the moving parts. The agitation caused by the movement of the carousel has a detrimental effect on the repeatability of the assay.
One additional characteristic of the microplate reader is that all the samples must be prepared at one time. The tests are not independently initiated.